Anti-tie2 antibody and use thereof
A TIE-2 and antibody technology, applied in the direction of antibodies, antibody medical components, applications, etc., can solve the problems of reduced drug accessibility and reduced therapeutic effect
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Embodiment 1
[0122] Example 1 Screening of Antibodies Binding to TIE2
[0123] A human primary ScFv (scFv) library disclosed in Korean Patent Laid-Open Publication No. 10-2008-0109417 was used to screen for antibodies binding to TIE2 and to prepare the library. Spread 100 μl of antigen (hTIE2-his: Sino Biological, 10700-H08H) on 96-well Ni at 2 μl / ml + plate and incubate overnight at 4°C. The next day, the antigen-coated plate was washed three times with 0.1% TBST, and then reacted with 200 μl of 2% BSA blocking buffer for 2 hours at room temperature. Add 50 μl of XL1-Blue stock solution to 2 ml of 2xYT-TET (tetracycline 10 μg / ml) growth medium, and incubate at 37°C at 200 rpm for 2 hours, then add 13 ml of 2xYT-TET growth medium, and Incubate until OD 600 reach 0.5. After blocking for 2 hours, the wells were washed 3 times with 1XPBS. The phage library group was combined with each washed well, the phage library was mixed with 4% BSA in equal amounts, and then 200 µl of the mixture wa...
Embodiment 2
[0124] Example 2 Screening of monoclonal ScFv phage that specifically binds to TIE2 and neutralizes the binding to TIE2 (combination ELISA)
[0125] After completing the panning process, the final circular cell stock solution was diluted to form 200 to 500 colonies, plated on CM agar plates, and then incubated overnight at 37 °C. The next day, when the colony grows, add 200 μl of 2xYT medium (CM 34 μg / ml+1% glucose) into a 96-well deep-well plate, insert one colony into each well, and then place the plate at 37°C and 3,000rpm Incubate overnight. The next day, spread 200 μl of 2xYT medium (CM 34 μg / ml+1% glucose) on a fresh 96-well deep-well plate, inject 20 ml of the cells grown the day before into each well, and incubate at 37°C and 3,000 Grow for 1 hour and 10 minutes at rpm. Store the remaining cells in 100 μl of 50% glycerol at -70 °C. While the cells were growing, mix 1 μl of helper phage with 19 μl of 2xYT medium, inject 20 μl of the resulting mixture into each well, ...
Embodiment 3
[0141] Example 3 Selection of antibodies with binding ability to cells expressing TIE2
[0142] To determine binding to TIE2-expressing cells, binding to self-established human / mouse TIE2-overexpressing CHO-K1 cell lines (hTIE2 / CHO-K1, mTIE2 / CHO-K1) was measured using flow cytometry. Each cell line was maintained and cultured, hTIE2 CHO-K1 was washed with PBS, 5 mM EDTA was added thereto to obtain a cell suspension, and each cell was recovered by centrifugation. Prepare 5×10 cells in FACS-specific buffer (PBS containing 2% FBS, 0.05% sodium azide) 6 cells / mL of cell suspension, and dispense 100 μL of cell suspension into each tube. Add 100 µL of 2 µg / mL clones to each tube and let stand at 4 °C for 30 min to induce cell binding. The cells were washed with 2 mL of FACS buffer, 100 μL of secondary antibody dilution (1 / 500, anti-human IgG-Fc polyclonal antibody fragment conjugated with PE, #A80-248PE, BethylLaboratories Inc.US) was added to it, and the resulting The mixture wa...
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